In addition to the well-known roles in development and maintenance of normal sexual and reproductive function, estrogen exerts a vast range of biological effects in nonclassical endocrine organ systems, such as cardiovascular system and liver. Bile acids are metabolites of cholesterol and have been identified as a class of signaling molecules for nuclear receptor farnesoid X receptor (FXR). Activation of FXR by bile acids regulates a myriad of FXR target genes involving in maintenance of bile acids, cholesterol, lipids and glucose homeostasis. Bile acid salt export pump (BSEP) positively regulated by bile acids through activating FXR plays a critical role in maintaining bile acid homeostasis in the liver and bile as a canalicular bile acid effluxer. Among diseases resulted from imbalance of bile acid levels, intrahepatic cholestasis of pregnancy (ICP) and gallstone disease are both associated with sex hormone estrogen. Although multiple risk factors are linked to those disorders, studies have demonstrated that estrogen plays a key role in the induction of the diseases. Both estrogen-induced cholestasis and gallstone disease have been reproduced in animal model. However, our understanding on the underlying mechanisms is far from complete. In our preliminary study, we found that estrogen repressed human BSEP expression in vitro and in vivo, and the repression was mediated by estrogen receptor 1 (ER1) through physically interacting with FXR, indicating a crosstalk between estrogen/ER1 and bile acids/FXR signaling pathway. The central hypothesis to be tested is that down-regulation of BSEP expression by estrogen is mediated through a novel nonclassical transrepression pathway, a direct interaction between ER1 and FXR and as a consequence is a common risk factor for ICP and gallstone disease. Two specific aims are proposed to test the hypothesis. Specific Aim 1 is to determine the mechanisms for estrogen-mediated repression of BSEP expression by the following three approaches: (1) the physical interaction between ER1 and FXR will be firmly established in vitro and in vivo; (2) altered recruitment of FXR and ER1 coregulators to the BSEP promoter by estrogen will be determined; and (3) the region in ER1 directly interacting with FXR will be mapped. Specific Aim 2 is to investigate estrogen-mediated BSEP repression, hepatic and biliary pathological consequences in vivo with the following two approaches: (1) the dynamics of human BSEP transactivation in vivo in mice during the entire pregnancy will be determined with the Living Imaging technology; and (2) the etiological role of estrogen-mediated BSEP repression in estrogen-induced cholestasis and gallstone disease will be investigated in wt and ER1(-/-) mice. Upon completion of the proposed study, we will have firmly established a novel nonclassical estrogen transrepression pathway through a direct interaction between ER1 and FXR, revealed mechanistic insights into such crosstalk between the estrogen/ER1 and bile acids/FXR signaling pathway, and established a common etiological role of BSEP repression in estrogen-induced cholestasis and gallstone disease. The findings will open a new field to advance our understanding on the diverse biological and pathological activities associated with estrogens through investigating estrogen's effects on various FXR target genes. Equally significant, the findings will advance our understanding on the mechanisms and pathogenesis of estrogen-induced ICP and gallstone disease. For the first time, the results will establish a common etiological link between the two distinct but related diseases, and finally, the findings will provide a molecular basis for developing therapeutic agents to treat or prevent those disorders by modulating BSEP expression and ER1/FXR signaling pathways. PUBLIC HEALTH RELEVANCE: Bile acids are synthesized in the liver and removed from the liver to bile by the bile salt export pump (BSEP). When BSEP production is damaged by female sex hormone estrogen, too much bile acids are accumulated in the liver, causing a liver disease called intrahepatic cholestasis of pregnancy (ICP), and too little bile acids are present in bile, potentially causing gallstone disease. Understanding the mechanisms and pathological consequences will provide a molecular basis to develop new drugs to treat or prevent those diseases.